Viruses are defined as minute infectious agents, not visible in the light microscope and unable to have independent metabolism and replicate outside the living cell. In order to multiply, viruses have to utilize living cell machinery. The living organism protects itself against viral infection by mounting nonspecific and specific (immune) defenses. The nonspecific mechanisms include phagocytosis, mechanical and chemical barriers, elevation of body temperature, inflammation and viral interference exerted by compounds such as interferons.
Phagocytosis in its primitive form is nonspecific, but it can be also viewed upon as a precursor of the immune defense. Until now phagocytosis seems to be the only defense of single cell organisms, such as protozoa against bacteria and viral infection. Whereas, it is undoubtedly successful as the defense mechanism of protozoa against bacteria, it is not sufficient to protect against viruses. Many different viruses will take advantage from being engulfed by phagocyte cells because this provides them an easy entry to the cell and they can then multiply in the phagocytes.
The obvious question to be asked is how unicellular organisms, such as protozoa, survived and were not eliminated by viruses over millions of years. It is postulated that protozoa possess a biochemical defense against viruses, which allows them to change the code governing reactions inside the cell and establish an environment hostile to virus replication. Two of such mechanisms have been found in protozoa already: deviation from the genetic code and RNA editing. According to data published by others, certain protozoa use stop codon TAG for incorporation of glutamine in the polypeptide chain (Preer, J. R. et al., Nature 314:188 (1985) and Caron, F. et al., Nature 314:185 (1988)). This allows the protein synthesis to continue through incorporation of glutamine instead of stopping at a stop codon. Such change will not allow viruses to produce correct proteins because the genetic code in viruses and protozoa will have different meaning.
RNA editing has been described in various protozoa and allows a change from cytosine to uracil in RNA (Powell, L. M. et al., Cell 50:831 (1987), Feagin, J. E. et al., Cell 53:413 (1988) and Simpson, L. et al., Cell 57:355 (1989)). This will result in stop codons UAG and UAA, instead of CAG and CAA, which are responsible for incorporation of glutamine. The outcome is production of different proteins than required by the viral genetic code.
As an additional response to viral infection, certain protozoa will multiply more rapidly and will form an entire colony instead of a single cell. Under such circumstances even if some cells die due to infection by the virus, the rest of the colony will survive.
To summarize, the defense of certain protozoa against viruses increases reliance on glutamine necessary for protein synthesis and stimulation of reproduction. The persistence of such mechanism in a higher organism may result in abnormal and uncontrolled cell growth leading to neoplastic growth.
As a possible defense mechanism, the multicellular organism can modify DNA bases. During the course of cellular differentiation, the tendency exists to eliminate methylated cytosine residues similar to the process of elimination of 5-methylcytosine in the course of evolution (Bird, A. P., Trends Genet. 3:342 (1987)). The final result is that cytosine is converted into thymine. This may decrease the rate of replication of DNA viruses which maintain CG base content higher than mature differentiated cells (Tooze, J., ed. The DNA Tumor Viruses, Molecular Biology of Tumor Viruses, 2d ed., Cold Spring Harbor, N.Y. 1981) and Baer, R. et al., Nature 310:207 (1984)). The disturbance of the differentiation process can delay modification of cytosine into thymine, create a greater reliance on glutamine because of persistence of CAG and CAA glutamine codons and allow the virus to replicate. Contrary to DNA viruses, the rate of evolutionary divergence of RNA viruses is very rapid which results in similar CG content in viral as well as host genome (Holland, J. et al., Science 215:1577 (1982) and Steinhauer, D. A. et al., J. Annu. Rev. Microbiol. 41:409 (1987)).
It is definitely advantageous for viruses to maintain host cells undifferentiated for as long as possible. Once the cell becomes terminally differentiated, the replication of certain viruses such as HIV, Herpes simplex and Epstein Barr virus may stop (Cullen, B. R. et al., Cell 58:423 (1989) and Garcia-Blanco, M. A. et al., Science 254:815 (1991)). On the other hand, undifferentiated cells require more glutamine. Induction of differentiation in the cells infected with virus, through inhibition of incorporation of glutamine may offer a chance to control the disease.
Without wishing to bound to any proposed theory, the present inventor postulates that the human body possesses a Biochemical Defense System (BDS) (Burzynski, S. R., Internat. J. Exp. Clin. Chemother. 2:63 (1989) and Burzynski, S. R., 17th Internat. Cong. Chemother., Berlin (1991)). This system parallels the immune defense, but protects the organism against the enemy within the body. The main purpose is no longer the defense against the micro-organism, but defense against defective cells. Such defective cells may occur as the result of viral infection. Chemical components of this biochemical defense system are peptides, amino acid derivatives and organic acids defined as antineoplastons (Burzynski, S. R., Physiol. Chem. Phys. 8:275 (1976) and Burzynski, S. R., U.S. Pat. No. 4,470,970). The mechanism of defense is based not on destruction, but on the reprogramming of defective cells through induction of differentiation.
The research on antineoplastons began in Poland in 1967 (Burzynski, S. R., Experientia 25:490 (1969) and Burzynski, S. R., Drugs Exptl. Clin. Res. Suppl. 1 12:1 (1986)). Initially, the work concentrated on the isolation of peptides which exist in the blood of healthy people and are deficient in cancer patients. Due to the small amount of raw material available for the study, in the following years, antineoplastons were isolated from urine instead of blood. In 1980 the structure of the first antineoplaston was identified and reproduced synthetically (Burzynski, S. R. et al., Proc. 13th Internat. Cong. Chemother., Vienna, Austria 17, P.S. 12. 4. 11-4).
Antineoplastons are divided into two groups. One group contains compounds which have a wide spectrum of activity and includes Antineoplaston A1, A2, A3, A4, A5, A10, AS2-1, AS2-5. Antineoplastons A1, A2, A3, A4 and A5 contain peptides isolated from urine and Antineoplaston A10, AS2-1 and AS2-5 are the synthetic products. See e.g. U.S. Pat. Nos. 4,470,970, 4,558,057 and 4,559,325. In addition to the first group, there are antineoplastons that are active against a single specific type of neoplasm, such as Antineoplaston H, L and O. Antineoplaston A10 is the first active ingredient isolated and reproduced by synthesis. Acid hydrolysis of Antineoplaston A10 initially produces phenylacetylglutamine and phenylacetylisoglutamine. When hydrolysis is carried further, the products of reaction include phenylacetic acid, glutamic acid and ammonia. The sodium salt of phenylacetylglutamine was named Antineoplaston AS2-5 and the mixture of the sodium salts of phenylacetylglutamine and phenylacetic acid in the ratio of 1:4 was named Antineoplaston AS2-1 (AS2-1) (Burzynski, S. R. et al., Drugs Exptl. Clin. Res. Suppl. 1, 12:11 (1986)).
According to the present inventor, AS2-1 seems to induce differentiation by reducing the level of glutamine in cells and substituting glutamine with phenylacetylglutamine. Relative excess of glutamine is essential for entering S-phase of cell cycle (Zetterberg, A. et al., Cell Physiol. Chem. 108:365 (1981)). In Swiss 3T3 cells cultured and starved to quiescence, a relative excess of glutamine is necessary for approximately seven hours from the end of G.sub.o to the beginning of S phase (Zetterberg, A. et al., Cell Physiol. Chem. 108:365 (1981)).
The availability of glutamine for cells in the human organism is regulated through the well-known reaction of the conjugation of glutamine with phenylacetic acid to phenylacetylglutamine (Thierfelder, H. et al., Z. Physiol. Chem. 94:1 (1915)). Phenylacetic acid is produced in substantial amounts in the human body and over 90% is bound with glutamine to form phenylacetylglutamine (Seakins, J. W. T., Clin. Chem. Acta. 35:121 (1971)). The type of amino acid conjugated with phenylacetic acid is different for different animals and is correlated with their evolutionary status. Conjugation of glutamine seems to be specific for humans and old world monkeys (James, M. O. et al., Proc. R. Soc. Lond. B. 182:25 (1972)). Systemic administration of AS2-1 to a patient produces a relative deficiency of glutamine and introduces phenylacetyglutamine which competes with glutamine.